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Although estuarine diatoms have a wide range of salt tolerance, they are often severely stressed by elevated salt concentrations. It remains poorly understood how estuarine diatoms maintain ionic homeostasis under high-salinity conditions. Using a scanning ion-selective electrode technique, this study determined the fluxes of H+, Na+, and K+ involved in the acclimatization of the estuarine diatom Coscinodiscus centralis Ehrenberg after an elevation in salinity from 15 psu to 35 psu. The C. centralis cells exhibited marked H+ effluxes after a transient treatment (TT, 30 min) and short-term treatment (ST, 24 h). However, a drastic shift of H+ efflux toward an influx was induced in the long-term treatment (LT, 10 days). The Na+ flux under TT, ST, and LT salinity conditions was found to accelerate the Na+ efflux. More pronounced effects were observed under the ST and LT salinity conditions compared to the TT salinity condition. The K+ influx showed a significant increase under the LT salinity condition. However, the salinity-induced Na+/H+ exchange in the estuarine diatom was inhibited by amiloride and sodium orthovanadate. These results indicate that the Na+ extrusion in salt-stressed cells is mainly the result of an active Na+/H+ antiport across the plasma membrane. The pattern of ion fluxes under the TT and ST salinity conditions were different from those under the LT salinity conditions, suggesting an incomplete regulation of the acclimation process in the estuarine diatom under short-term salinity stress.

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Molecular and physiological analyses in ionoregulatory organs (e.g., adult gills and embryonic skin) are essential for studying fish ion regulation. Recent progress in the molecular physiology of fish ion regulation was mostly obtained in embryonic skin; however, studies of ion regulation in adult gills are still elusive and limited because there are no direct methods for in vivo functional assays in the gills. The present study applied the scanning ion-selective electrode technique (SIET) in adult gills to investigate branchial H+-excreting functions in vivo. We removed the opercula from zebrafish and then performed long-term acid acclimation experiments. The results of Western blot and immunofluorescence showed that the protein expression of H+-ATPase (HA) and the number of H+-ATPase-rich ionocytes were increased under acidic situations. The SIET results proved that the H+ excretion capacity is indeed enhanced in the gills acclimated to acidic water. In addition, both HA and Na+/H+ exchanger (Nhe) inhibitors suppressed the branchial H+ excretion capacity, suggesting that H+ is excreted in association with HA and Nhe in zebrafish gills. These results demonstrate that SIET is effective for in vivo detection in fish gills, representing a breakthrough approach for studying the molecular physiology of fish ion regulation.

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Transporting epithelia are tissues that specialize in the directional movements of ions and water and are typically either secretory or reabsorptive. Recent work on the Malpighian tubule of larval lepidopterans (caterpillars) demonstrated that the distal ileac plexus segment of this epithelium is capable of rapidly switching between ion secretion and reabsorption. Subsequent transcriptomic studies suggested expression of voltage-gated ion channels in the lepidopteran MTs (which are not contractile and not innervated). The present study shows that isolated MTs of larval Trichoplusia ni express α1, ß2, and α2δ4 subunits of voltage-gated Ca2+ channel CaV1 and that pan-CaVα immunoreactivity is present in the apical and basolateral membranes of the principal cells. Basolateral membrane potential (Vbl) in isolated MTs of larval Trichoplusia ni was influenced by CaV1 functioning; pharmacological inhibition of CaV1 reversed Vbl from inside-negative to inside-positive, and also reduced transepithelial potential (Vte), lowered [Ca2+]i and reversed the direction of K+ transport from secretion to reabsorption. Thus, our findings indicate that a functional CaV1 channel is necessary for constitutive K+ secretion observed in isolated preparations of lepidopteran MTs. Lastly, Vte and Vbl of isolated MTs were influenced by changes in bathing saline [K+]. Our findings suggest that epithelia may rely on CaV channels to enable robust ion secretion and downregulation of CaV channels, together with other transcriptional changes, enables ion reabsorption.

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The elasmoid scales of anadromous sea trout Salmo trutta L. represent a significant internal reservoir of Ca2+ . Although more is known about long-term remodelling of scales in response to calciotropic challenges encountered during smoltification and migration, very little is known about the contribution made by scales to the short-term, minute-to-minute regulation of Ca2+ homeostasis in the extracellular fluid (ECF) during these phases of the life cycle. This gap in the knowledge is partly due to the technical challenges involved in measuring small Ca2+ fluxes around the scales of live fish in real time. Here, this study describes exfoliating, mounting and culturing scales and their resident cells from parr, smolt and adult sea trout from a freshwater environment, as well as from adult sea trout caught in sea or brackish water. All the scales were then examined using an extracellular, non-invasive, surface-scanning Ca2+ -sensitive microelectrode. The authors quantified the Ca2+ fluxes, in the absence of any systemic or local regulators, into and out of scales on both the episquamal and hyposquamal sides under different extracellular calcemic challenges set to mimic a variety of ECF-Ca2+ concentrations. Scales from the life-cycle stages as well as from adult fish taken from sea, brackish or fresh water all showed a consistent efflux or influx of Ca2+ under hypo- or hypercalcemic conditions, respectively. What were considered to be isocalcemic conditions resulted in minimal flux of Ca2+ in either direction, or in the case of adult scales, a consistent but small influx. Indeed, adult scales appeared to display the largest flux densities in either direction. These new data extend the current understanding of the role played by fish scales in the short-term, minute-to-minute homeostatic regulation of ECF-Ca2+ concentration, and are similar to those recently reported from zebrafish Danio rerio scales. This suggests that this short-term regulatory response might be a common feature of teleost scales.

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Pyrokinins are structurally related insect neuropeptides, characterized by their myotropic, pheromonotropic and melanotropic roles in some insects, but their function is unclear in blood-feeding arthropods. In the present study, we functionally characterized the pyrokinin-1 and pyrokinin-2 receptors (PK1-R and PK2-R, respectively), in the yellow fever mosquito, Aedes aegypti, using a heterologous cell system to characterize their selective and dose-responsive activation by members of two distinct pyrokinin subfamilies. We also assessed transcript-level expression of these receptors in adult organs and found the highest level of PK1-R transcript in the posterior hindgut (rectum) while PK2-R expression was enriched in the anterior hindgut (ileum) as well as in reproductive organs, suggesting these to be prominent target sites for their peptidergic ligands. In support of this, PRXa-like immunoreactivity (where X = V or L) was localized to innervation along the hindgut. Indeed, we identified a myoinhibitory role for a PK2 on the ileum where PK2-R transcript was enriched. However, although we found that PK1 did not influence myoactivity or Na+ transport in isolated recta, the PRXa-like immunolocalization terminating in close association to the rectal pads and the significant enrichment of PK1-R transcript in the rectum suggests this organ could be a target of PK1 signaling and may regulate the excretory system in this important disease vector species.

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Acidification of freshwater ecosystems is recognized as a global environmental problem. However, the influence of acidic water on the early stages of freshwater fish is still unclear. This study focused on the sublethal effects of acidic water on the lateral line system of zebrafish embryos. Zebrafish embryos were exposed to water at different pH values (pH 4, 5, 7, 9, and 10) for 96 (0-96 h post-fertilization (hpf)) and 48 h (48∼96 hpf). The survival rate, body length, and heart rate significantly decreased in pH 4-exposed embryos during the 96-h incubation. The number of lateral-line neuromasts and the size of otic vesicles/otoliths also decreased in pH 4-exposed embryos subjected to 96- and 48-h incubations. The number of neuromasts decreased in pH 5-exposed embryos during the 96-h incubation. Alkaline water (pH 9 and 10) did not influence embryonic development but suppressed the hatching process. The mechanotransducer channel-mediated Ca2+ influx was measured to reveal the function of lateral line hair cells. The Ca2+ influx of hair cells decreased in pH 5-exposed embryos subjected to the 48-h incubation, and both the number and Ca2+ influx of hair cells had decreased in pH 5-exposed embryos after 96 h of incubation. In addition, the number and function of hair cells were suppressed in H+-ATPase- or GCM2-knockdown embryos, which partially lost the ability to secrete acid into the ambient water. In conclusion, this study suggests that lateral line hair cells are sensitive to an acidic environment, and freshwater acidification could be a threat to the early stages of fishes.

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Springtails (Collembola) are ancient close relatives of the insects. The eversible vesicles are their unique paired transporting organs, which consist of an epithelium located inside a tube-like structure called the collophore on the first abdominal segment. The vesicles can be protruded out of the collophore and several lines of evidence indicate that they have a vital function in water uptake and ion balance. However, the amount of water absorbed by the vesicles and which other ions apart from Na+ are transported remain unknown. Using Orchesella cincta as a model, we developed protocols for two assays that enabled us to study water and ion movement across the eversible vesicles in whole living springtails. Using an inverse Ramsay assay we demonstrate that the eversible vesicles absorb water from a droplet applied onto their surface. Using the scanning ion-selective electrode technique (SIET), we show that the vesicles absorb Na+ and Cl- from the bathing medium, secrete NH4+, and both absorb and secrete K+ H+ is secreted at a low level in the anterior part and absorbed at the posterior part. We did not detect transport of Ca2+ at significant levels. The highest flux was the absorption of Cl-, and the magnitude of ion fluxes was significantly lower in fully hydrated springtails. Our data demonstrate that the eversible vesicles are a transporting epithelium functioning in osmo- and ionoregulation, nitrogenous waste excretion and probably also acid-base balance.

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Calcium ion concentration ([Ca2+]) in the systemic extracellular fluid, ECF-[Ca2+], is maintained around a genetically predetermined set-point, which combines the operational level of the kidney and bone/ECF interfaces. The ECF-[Ca2+] is maintained within a narrow oscillation range by the regulatory action of Parathyroid Hormone (PTH), Calcitonin, FGF-23, and 1,25(OH)2D3. This model implies two correction mechanisms, i.e. tubular Ca2+ reabsorption and osteoclast Ca2+ resorption. Although their alterations have an effect on the ECF-[Ca2+] maintenance, they cannot fully account for rapid correction of the continuing perturbations of plasma [Ca2+], which occur daily in life. The existence of Ca2+ fluxes at quiescent bone surfaces fulfills the role of a short-term error correction mechanism in Ca2+ homeostasis. To explore the hypothesis that PTH regulates the cell system responsible for the fast Ca2+ fluxes at the bone/ECF interface, we have performed direct real-time measurements of Ca2+ fluxes at the surface of ex-vivo metatarsal bones maintained in physiological conditions mimicking ECF, and exposed to PTH. To further characterize whether the PTH receptor on osteocytes is a critical component of the minute-to-minute ECF-[Ca2+] regulation, metatarsal bones from mice lacking the PTH receptor in these cells were tested ex vivo for rapid Ca2+ exchange. We performed direct real-time measurements of Ca2+ fluxes and concentration gradients by a scanning ion-selective electrode technique (SIET). To validate ex vivo measurements, we also evaluated acute calcemic response to PTH in vivo in mice lacking PTH receptors in osteocytes vs littermate controls. Our data demonstrated that Ca2+ fluxes at the bone-ECF interface in excised bones as well as acute calcemic response in the short-term were unaffected by PTH exposure and its signaling through its receptor in osteocytes. Rapid minute-to-minute regulation of the ECF-[Ca2+] was found to be independent of PTH actions on osteocytes. Similarly, mice lacking PTH receptor in osteocytes, responded to PTH challenge with similar calcemic increases.

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Chironomids are often one of the dominant organisms in significantly polluted freshwater. Many invertebrate studies have characterized whole-organism mechanisms of toxicity, for example, assessing cadmium (Cd) uptake via calcium (Ca) channels. However, with the use of the scanning ion-selective electrode technique and an innovative Cd-selective microelectrode, we analyze this relationship at the organ level using a realistic concentration of Cd and Ca in the hemolymph (blood). Generally, Cd fluxes follow the same directional pattern as Ca, although Ca fluxes are approximately 5 times higher than those of Cd. These results correlate well with previous studies indicating that chironomids have a higher affinity for Ca over Cd, which affords them tolerance to Cd toxicity. When saline Ca concentration was increased to 10 times physiological levels, Cd fluxes from the gut lumen into the cells of the midgut regions were reduced by 50 to 80%. Transport of Cd from hemolymph to tissue for the posterior midgut, Malpighian tubule, and proximal ceca was also reduced by approximately 50%. The present results indicate that Cd fluxes into or across the gut and Malpighian tubules are reduced by high Ca, suggesting that Cd may be transported in some cells by similar mechanisms. However, Cd was actively excreted at the anal papillae after a 48-h waterborne exposure to Cd, but this process was independent of Ca and instead may involve a P-glycoprotein-related pump to detoxify Cd. Environ Toxicol Chem 2018;37:2542-2549. © 2018 SETAC.

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The calcium-sensing receptor (CaSR) is an extracellular Ca2+ sensor that plays a critical role in maintaining Ca2+ homeostasis in several organs, including the parathyroid gland and kidneys. In this study, through in situ hybridization, the expression of CaSR mRNA was found in the neuromasts of zebrafish larvae. Immunohistochemistry further demonstrated that the CaSR protein was present in neuromast hair cell stereocilia and basolateral membranes. Based on the expression and subcellular localization of the CaSR in hair cells, we hypothesized that the CaSR is expressed in zebrafish lateral-line hair cells to regulate mechanotransducer (MET)-channel-mediated Ca2+ entry. Using the scanning ion-selective electrode technique, MET-channel-mediated Ca2+ influx at the stereocilia of hair cells was measured in intact larvae. Ca2+ influx was suppressed after larvae were pretreated with a CaSR activator (R-568) or high-Ca2+ (HCa) medium. Gene knockdown by using morpholino oligonucleotides decreased CaSR expression in hair cells and eliminated the effects of R-568 and HCa on Ca2+ influx. In addition, we found that treatment with R-568 attenuated neomycin-induced hair cell death. This study is the first to demonstrate that the CaSR is involved in mechanotransduction in zebrafish hair cells.

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Polyamines play a regulatory role in eukaryotic cell growth and morphogenesis. Despite many molecular advances, the underlying mechanism of action remains unclear. Here, we investigate a mechanism by which spermine affects the morphogenesis of a dimorphic fungal model of emerging relevance in plant interactions, Yarrowia lipolytica, through the recruitment of a phytohormone-like pathway involving activation of the plasma membrane P-type H+-ATPase. Morphological transition was followed microscopically, and the H+-ATPase activity was analyzed in isolated membrane vesicles. Proton flux and acidification were directly probed at living cell surfaces by a non-invasive selective ion electrode technique. Spermine and indol-3-acetic acid (IAA) induced the yeast-hypha transition, influencing the colony architecture. Spermine induced H+-ATPase activity and H+ efflux in living cells correlating with yeast-hypha dynamics. Pharmacological inhibition of spermine and IAA pathways prevented the physio-morphological responses, and indicated that spermine could act upstream of the IAA pathway. This study provides the first compelling evidence on the fungal morphogenesis and colony development as modulated by a spermine-induced acid growth mechanism analogous to that previously postulated for the multicellular growth regulation of plants.

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Cadmium (Cd) is a toxic element that poses a great threat to human health, while silicon (Si) is a beneficial element and has been shown to have a mitigation effect on plants under Cd toxicity. However, the mechanisms underlying the role of Si in alleviating Cd toxicity are still poorly understood in wheat. Therefore, growth status, photosynthesis parameters, root morphology, antioxidant system, and Cd2+ uptake and flux under Cd toxicity were studied through hydroponic experiment, aiming to explore the mitigation of Si on Cd toxicity in wheat seedlings. The results showed that Si supply improved plant biomass as well as photosynthetic but had little effects on root morphology of seedlings under Cd stress. Si addition decreased Cd contents both in shoots and roots. In situ measurements of Cd2+ flux showed that Si significantly inhibited the net Cd2+ influx in roots of wheat. Si also mitigated the oxidative stress in wheat leaves by decreasing malondialdialdehyde (MDA) and hydrogen peroxide (H2O2) contents as well as by increasing superoxide dismutase (SOD) and guaiacol peroxidase (POD) activity. Overall, the results revealed that Si could alleviate Cd toxicity in wheat seedlings by improving plant growth and antioxidant capacity and by decreasing Cd uptake and lipid peroxidation.

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Wheat is one of several cereals that is capable of accumulating higher amounts of Cd in plant tissues. It is important to understand the Cd2+ transport processes in roots that result in excess Cd accumulation. Traditional destructive technologies have limited capabilities in analyzing root samples due to methodological limitations, and sometimes may result in false conclusions. The mechanisms of Cd2+ uptake into the roots of wheat seedlings (Triticum aestivum L.) were investigated by assessing the impact of various inhibitors and channel blockers on Cd accumulation as well as the real-time net Cd2+ flux at roots with the non-destructive scanning ion-selective electrode technique. The P-type ATPase inhibitor Na3VO4 (500 µM) had little effect on Cd uptake (p < 0.05) and the kinetics of transport in the root of wheat, suggesting that Cd2+ uptake into wheat root cells is not directly dependent on H+ gradients. While, the uncoupler 2,4-dinitrophenol significantly limited Cd2+ uptake (p < 0.05) and transport kinetics in the root of wheat, suggesting the existence of metabolic mediation in the Cd2+ uptake process by wheat. The Cd content at the whole-plant level in wheat was significantly (p < 0.05) decreased upon pretreatment with the Ca2+ channel blockers La3+ or Gd3+ and Verapamil, but not in case of pretreatment with the K+ channel blocker tetraethylammonium (TEA). In addition, the inhibitors of the Ca2+ channel, as well as high concentrations of Ca2+, reduced the real-time net Cd2+ fluxes at the root surface in SIET experiments. These results indicate that Cd2+ moves across the plasma lemma of the wheat root via Ca2+ channels. In addition, our results suggested a role for protein synthesis in mediating Cd2+ uptake and transport by wheat.

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The scanning ion-selective electrode technique (SIET) was utilized for the first time in Locusta migratoria to characterize K(+) transport along the digestive tract and to determine the effect of two locust FGLamide allatostatins (FGLa/ASTs) on K(+) transport: a previously sequenced FGLa/AST from Schistocerca gregaria (Scg-AST-6; ARPYSFGL-NH2) and a newly sequenced FGLa/AST from L. migratoria (Locmi-FGLa/AST-2; LPVYNFGL-NH2). Regional differences in K(+) fluxes along the gut were evident, where K(+) efflux in vitro (or absorption into the hemolymph in vivo) was greatest at the anterior ileum, and lowest at the colon. Ileal K(+) efflux was inhibited by both Scg-AST-6 and Locmi-FGLa/AST-2, with maximal inhibition at 10(-10) and 10(-11) mol l(-1), respectively. Both FGLa/ASTs also inhibited cAMP-stimulated K(+) efflux from the ileum. Locmi-FGLa/AST-2 also inhibited efflux of water across the ileum. Locusts are terrestrial insects living in dry climates, risking desiccation and making water conservation a necessity. The results suggest that FGLa/ASTs may be acting as diuretics by increasing K(+) excretion and therefore increasing water excretion. Thus it is likely that FGLa/ASTs are involved in the control of hemolymph water and ion levels during feeding and digestion, to help the locust deal with the excess K(+) load (and subsequently fluid) when the meal is processed.

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